5-(5&#39;-barbiturilidene)-4-oxothiazolidine-2-imino hci salt used as an antimicrobial agent

ABSTRACT

1. A METHOD OF KILLING OR INHIBITING THE GROWTH OF MICROORGANISMS SELECTED FROM THE GROUP CONSISTING OF FUNGI, GRAM POSITIVE BACTERIA AND GRAM NEGATIVE BACTERIA WHICH COMPRISES CONTACTING SAID MICROORGANISMS WITH 5-(5&#39;&#39;-BARBITURILIDENE)4=OXOTHIAZOLIDINE-2-IMINO HCL SALT IN AN AMOUNT EFFECTIVE TO KILL OR INHIBIT THE GROWTH OF SAID MICROORGANISM.

United States Patent 3,849,560 -(5'-BARBITURILIDENE) 4 OXOTHIAZOLIDINE- Z-IMINO HCl SALT USED AS AN ANTIMICRO- BIAL AGENT Al F. Kerst, 5470 W. Geddes and John D. Douros, Jr., 6855 S. Depew, both of Littleton, Colo. 80123, and Milan Brokl, 1633 Cherry St., Denver, Colo. 80122 No Drawing. Continuation-impart of abandoned application Ser. No. 180,923, Sept. 15, 1971. This application Dec. 1, 1972, Ser. No. 311,075

Int. Cl. A01n 9/12 US. Cl. 424-254 50 Claims ABSTRACT OF THE DISCLOSURE 5-(5-barbituri1idene)-4-oxothiazolidine 2 imino HCl salt can be used to inhibit and/or prevent growth of undesirable bacteria, fungi, yeast, and other microorganisms. This invention is particularly concerned with the bacteriostatic and bactericidal properties of S-(Sbarbiturilidene-4-oxothiazolidine-Z-imino HCl salt compound against species of Mycobacterium, Salmonella, and Staphylococcus. This compound also combats the growth of certain undesirable aquatic plants such as Duckweed and Anacharis.

BACKGROUND OF THE INVENTION This application is a continuation-in-part of our application Ser. No. 180,923, filed Sept. 15, 1971, now abandoned. While there is no dearth of barbituric microbial growth inhibitors existing today, the antimicrobial properties of 5-(5-barbiturilidene)-4-oxothiazolid'ine-2-imino HCI salt have not been previously discovered. Furthermore few of the commercially available barbituric growth inhibitors offer the activity of 5-(5-barbiturilidene)-4- oxothiazolidine-Z-imino HCl salt against such a broad spectrum of bacteria, fungi, yeast, et cetera. This broad activity is often desirable since the inhibition of one microorganism species or group of species may create an imbalance which often results in the rampant growth of other deleterious microorganisms.

SUMMARY OF THE INVENTION According to the present invention, it has been found that the newly discovered compound 5-(5'-barbiturilidene)-4-oxothiazolidine-2-imino HCl salt is a very effective antimicrobial agent. Our procedure for preparing this compound proceeds according to the following.

EXAMPLE 1 One-tenth mole (16.0 g.) of alloxan monohydrate and 0.125 mole of (15.5 g.) of pseudothiohydantoin hydrochloride prepared according to the methods taught in Org. Synth., Coll. vol. III, p. 751) are refluxed in 200 ml. of absol. (methanol) on a water bath for three hours. After this reaction time, the solution which is a deep yellow is concentrated through evaporation in vacuo to about A of the original volume and the concentrate is allowed tostand under refrigeration for about 12 hours.

' Yellow crystals are formed on the bottom and walls of the breaker as microscopic needles and are separated on a sintered glass funnel, washed with small portions of water and acetone, and dried in an evacuated dessicator. About 18.4 g. (67.4% of theoretical) of the compound (melt pt. 360 C.) are formed.

EXAMPLE 2 The reaction of example 1 is repeated using the reaction conditions and the same molar ratios of alloxan monohydrate and pseudothiohydantoin and replacing the methanol solvent with solvents selected from the 3,849,560 Patented Nov. 1?) 1974 DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples illustrate the antimicrobial qualities of 5-(5-barbiturilidene) 4 oxothiazolidine-2- imino HCl salt and described how these antimicrobial qualtities may be utilized in various phases of agriculture, animal husbandry, and pharmacology.

EXAMPLE 3.-Antibacterial and Antiyeast Activity The in vitro effectiveness of 5-(5'-barbiturilidene)-4- oxothiazolidine-Z-imino HCl salt against bacteria and yeast species is established in the following manner. One loopful of each of the investigated bacteria or yeast is transferred from agar slants to 10 ml. of trypticase soy broth and incubated at 37 for 18 hours. At the end of this period, the bacteria or yeast is seeded into the same medium (LS-2% agar) in which the original inoculum is prepared. The bacteria are then seeded at 1 ml. of inoculum per 250 ml. of medium, which is equivalent to at least 1X 10 cells/ml. determined by dilution platecount or nephelome'ter readings which has previously been verified by counts. The resultant mixtures are poured into heat-resistant sterile petri dishes at a temperature of 45 C. Analytical filter paper discs 1.2 cm. in diameter are used for the agar diffusion technique. 'Each disc is saturated with 0.08 ml. of the solubilized 5-(5barbiturilidene)- 4-oxothiazolidine-2-imino HCl salt compound ,ug./ disc) and placed on the surface of the hardened agar. The plates are incubated at 37 C. for 18 hours. The activity of the 5'-(barbiturilidene)-4-oxothiazolidine-2-imino HCl salt compound is established by measuring the zone of inhibition in centimeters. Untreated control plates are used as a basis for comparison and these exhibit a profuse growth of bacteria. The results of these tests are as follows:

Shigella boydii:

A'ICC No. 9905 Shigella sonnei, MMV 6654 Shtgella dyeenteriae Type 2, MMV 6673. Salmonella panama, ATCO No. 7378... Salmonella paratyphi, A'ICC N0. 9281.

Salmonella pnllorum. ATOC No. 10398 Salmonella derby, ATOC N0. 6960 Salmonella gallinarium, ATCO No. 9184- Salmonella typhimarium, SR-ll Salmonella typhosa, ATOC No. 19043.. Nezsserz'a gonorrhoeae, A'ICC N 0. 19424 Neisseria meningitides:

ATCC No. available on request Vibrio cholerae, A'ICC No. 14035.. Proteus vnlgarls, A'ICC No. 4984.--. Erwinia caralovora, ATCC No. 495. Mycobaclerium butyricum, ATCC No. 11314 Mycobacteriam fortuitam, Debos, ATCC No. 4243-. Mycobacteriam avium, ATCC No. 19421 Mycobacterium tuberculosis, Va. hominis Mz cobacterlnm phlei, ATCC No. 11782, phage host- Klebszella pneumoniae Micrococcua tetraqena, ATCC No. 10875 TABLEContinued Zone of inhibition in cen- Gram positive and gram negative bacteria timeters Micrococcus melitemis, ATCC No. 19399- 2. 5 Micrococcus lysodeikticue, ATCC No. 4698 2. 7 Carynebacterium diphtheriae, ATCO No. 1 2. Corynebueterium hoemolyticum, ATCC No. 9345. 2. 0 Dlplococcus intracellularis 3. i Dzplococcus pneumoniae, A'ICC N o. 6303-.. 3. 4 Hemophilus hemclyticus, ATCC No. 10014 tr Hemophilus influenzae, ATCO No. 19418 tr Hemophilus parainfluenzae, ATCG No. 7901 tr Hemophilus suis, ATOO No. 19417 tr Hemophilua vaqinalis, ATCC No. 14018- tr Brucella abortus, ATCC No. 4315 tr Brucella melitensis ATCC No. 19396. tr Brucella suis, ATCC No. 4312 tr No'rE.tr=trace.

EXlAMPL'E 4.SECON'DARY SCREEN ANTIBAC- TERIAL ACTIVITY To further define the scope of this invention, secondary screening tests using the techniques described in the primary screening tests were also employed against selected bacteria genera at a concentration of SO/ g/disc. The results of these secondary screens are as follows:

Compound: 5-(5'-barbiturilidene)-4- oxothiazolidine-Z-imino HCl salt Microorganism Salmonella sp., ATCC No. 9120- Salmonella typhimurium miruglia, S Salmonella No. 45 MM Salmonella B/d, lVi Salmonella, MMU 66 Pseudomonas aer-uginosa:

A'ICC No. 10145 ATCC No. 8709..

XSTCC No. 12055. Pseudomonus maltophilia, G 107.. Pseudornonas K997 Pseudomonas K966 Proteus vulgcris.

ATCC No. 12454 Proteus mirabilis, ATCC No. 9961. Proteus morgauii, G951 Proteus mirabilis:

Comments: h=hazy zone; tr=trace.

This data indicates that 5-(5'-barbiturilidene)-4-oxothiazolidine-Z-imino HCI salt compound can be used to inhibit many important types of diseases. For example, it can be used against:

caused by various pseudomonads. Urinary miections are notable examples.

Erwinia cartovora Various species of Erwiniu attack commercial crops of carrots, tobacco, potatoes squash, et cetera.

Xanthomonosphaseoli Various species of Xanthomomzs cause a varietyoi diseases in plants such as sugar cane, rice, sugar beets, cotton, walnuts, wheat, rye, barley, beans, et cetera.

The demonstrated antibacterial activity of 5-(5'-barbiturilidene) 4 oxothiazolidine-Z-imino HCl salt compound against Staphylococcus aureus and Escherichia coli is of particular interest to the field of pharmacology since the disclosed activity indicates that this compound can be formulated as powders, selves, and ointments for administration in the treatment of burns and bacterially induced infiammations such as abscesses, dermatitis, rashes, and the like, particularly in domestic animals.

Although the precise mode of action whereby 5-(5'-barbiturilidene) 4 oXothiazolidine-2-imino HCl salt inhibits bacteria growth is not completely understood, it is believed that the 5-(5-barbiturilidene) 4 oxothiazolidine-Z-imino HCl salt compound of this invention may serve as a chemical antagonist; that is, as a chemical which competes with enzymes essential to the development of such bacteria. Since enzymes perform their catalytic function by virtue of their aflinity for their natural substrate; any compound resembling a substrate in its chemically critical aspect may also have an affinity for the enzymes. If this aflinity is great enough, the analog will displace the normal substrate from the enzyme and will prevent the growth reaction from taking place. It is believed that 5-(5'-barbiturilidene) 4 oxothiazolidine-Z- imino HCl salt has a chemical affinity for an essential site on one enzyme necessary for bacterial growth and life.

The 5 (Sbarbiturilidene) 4 oxothiazolidine-Z-imino HCl salt formulations of this invention can also contain other therapeutically valued supplements such as local anesthetics, irradiated oils, and other medicinal substances. When used for these or similar purposes, 5 (S'barbiturilidene) 4 oxothiazolidine 2 imino HCl salt may be incorporated in any therapeutically acceptable carrier such as oils, salves and ointments, together with adjuvauts comprising surface active agents, detergents, dispersing agents, stablizers and other modifiers which may facilitate the handling and application of the antibacterial material. In the case of the in vitro applications of the compositions of this invention, it is difiicult to predict with precision what in all cases will constitute a therapeutic dose even on a weight basis. Variable factors such as type, duration and severity of infection and mode of administration may be determining factors for the establishment of therapeutic doses.

Those skilled in the art will recognize that the above data indicates that the scope of this invention should not be limited to any particular disease species or to any particular type of animal or plant life. For example, the noted activity of 5 (Sbarbiturilidene) 4 oxothiazolidine-Z-imino HCl salt against Mycobacterium butyricum, Mycobacterium Tuberculosis var. hominis, and Mycobacterium avian indicates that this compound will also prove to be of value against such other Mycobacterium species as Mycobaterium bovis and Mycobacterium leprae.

EXAMPLE 5.ANTIFUNGAL ACTIVITY The antifungal activity of 5 (S'barbiturilidene)-4-oxothioazolidine 2 imino HCl salt compound is established by treating F usarium oxysporum, F usarium roseum, Rhizopus nigricans, Rhizopus stolonifer, Aspergillus niger and Alternaria solani test fungi in the following manner:

One loopful of each of the tested viable fungi cultures, spores and mycelia is transferred from an agar slant to an ml. portion of the nutrient broth composed of oatmeal agar, Czapeks, Sabouraud and deionized water to volume. The 80 ml. portion of the fungi and broth is then placed in a sterile shake flask (300 m1.) and the flask is placed on a rotary shaker for 96 to hours at room temperature. At the end of this incubation time period, 10 ml. of the liquid are homogenized and placed into another sterile shake flask (300 ml.) containing 80 ml. of the above nutrient broth and 60 ppm. of the inhibitor being evaluated. The flasks are placed on a rotary shaker operating at 240 r.p.m. at room temperature for 3 to 9 days.

After this second incubation time, the flasks are taken off and examined for visible fungal growth and mycelial weights are determined. Untreated controls are used as the basis of comparison and these displayed profuse fungal growth containing species of Fusarium, Rhizopus, Aspergillus and Alternaria. The results of these tests indicated that the 5 (Sbarbiturilidene) 4 oxothiazolidine-2- imino HCl salt compound of this invention imparts a substantial degree of inhibition of fungal growth at 60 p.p.m.

EXAMPLE 6.ANTIFUNGAL AND ANTIYEAST ACTIVITY To further define the antifungal activity of S-(S'barbiturilidene) 4 oxothiazolidine 2 imino HCl salt the seeded agar plates are prepared by transferring the cultures from slants washed with saline or phosphate buffer and then transferred to the surface of hardened Sabourand-Dextrose agar plates. Again, as in the case of Example 3, the S-(Sbarbiturilidene) 4 oxothiazolidine- 2-imino HCl salt is tested by impregnating filter paper discs (1.27 cm. in diameter) with 0.08 ml. of the solubilized 5 (Sbarbiturilidene) 4 oxothiazolidine-2-imino HCl salt compound (100 ,ag./disc) and placed on the surface of the hardened agar. The plates are incubated 2 at 30 C. for 18 hours. The activity of 5-(5'-barbiturilidene) 4 oxothiazolidine 2 imino HCl salt is established by measuring the zone of inhibition in centimeters. Untreated control plates are used as a basis for comparison and these inhibit a profuse growth of bacteria. The U results of these tests are as follows:

EXAMPLE 7.-SECONDARY FUNGAL SCREEN A secondary screen using the techniques of Example 6 produced the following results at the 5-(5barbiturilidene)- 4 oxothiazolidine 2 imino HCl salt concentrations indicated:

Concentration (Ag/disc) Microorganism Rhodotorula sp., Duke Rhizop'as stolonifer, ATCC No. 10404 Monascus parpurea, OU Fusarium rose'am, UFGC 1166 Fasarium oxysporum plus cubmse UFCC 1122 Scopalariopsis sp., OU. Aspergillus niger, SRI Aspergillas niger" Aspergz'llus sydowi, Aspergillus nidalans, ATCC N0. 100 Aspergillus flat/us, AICC N0. 9643 Aspergillus amstelodami, ATOC N Aspergt'llus fumiqatus Cephalosporiam acremrmium, A'ICC C'cphalosporium sp., 0U Phoma pigmentovora, A'ICC No. 12569- Paecilomyces varioti, ATCO No. 1114 Nigrospora sphaerica, ATCC No. 11687 Absidia spi'aosa, ATCC No. 6648..." Mucor racemosus, ATCC N0. 1216- Thamnidia'm elegans, NSC 8997-..- Phycomyces nitens, ATTC No. 9984.. Penicillium rubrum, ATCC No. 1052 Penicilliam notatwm, 0U Beauvaria bassia'na, MV 1341. Beauvaria tenella, MV 1919... Phtalophora verracosa, 0U. Cladasporium trichoides, 0U. Torula bergm', 0U Mtmosporum apiospermum, OU. Altemaria solam, ATCC No. 639..-.. Verticilliam albo-at1u'm, ATCC No. 108 Trichophyton mmtagrophytes, ATCC No. 9129. Trichophyton mentagrophyles, ATCC No. 8215- Trichophyton tonsurans, ATCC No. 10217.-. Pythiam arrhenomanes, ATTC No. 12531.. Helmiathosporiam oryzae, ATCC No. 11000 Comments: tr=trace.

It will also be recognized by those skilled in the art that other protectant, systemic and eradicant procedures may provide detection of other biological activities. Pathogens representative of Phycomycetes, Ascomycetes, Basidiomycetes and the Fungi Imperfecti may provide indices of other fungicidal activity. Additional pathogens and appropriate host plants may well afford other opportunities to further define the degree and spectrum of the activity disclosed in this invention. Since no firm rules of procedure can be laid down for the sequence of such evaluations or for the choice of pathogens, S-(S'barbiturilidene)-4-oxothiazolidine-2-imino HCl salt must be considered on the basis of its demonstrated performance in such primary evaluations and then progressively judged in subsequent studies. A wide range of pathogens, representative of economically important diseases, can be used to help define 5-(S'barbiturilidene)-4-oxothiazolidine-Z-imino HCl salt biological activity and to assure high degrees of success under field conditions. The following disease organisms, crops and reference standards may be used in such evaluations:

Reference Disease Disease organism compound Powdery mildew of Erysiphe cichoraceamm.. Maneb,

cucumbers. karathane. Leaf rust of wheat Puccim'a rubz'go-vera Do.

Do"-.. .do Plantvax. Rice blastdrsease Ptricularia oryzae. Blastidicin. Dgwry mildew of sugar Perzmospora schacm Karathane,

ee Dgwny mildew of lime Phytophthora phaseoli Do.

can. Bean rust Uromyces phaseoli var. Maneb.

, pica. Powdery mildew of wheat Erysiphe graminis Karathane. Powdery m ldew of apple- Podosphaera Zeucot1icha...- Do. Powdery mildew of roses. Sphaerotheca pamwsa var. Do.

rosae. Powdery mildew of Erg/siphe cichoracearum Do.

cantalope. Leaf spot of wheat Helminthosporium satiuam. Maneb. Early blight of tomato. Alternarz'a soltmi Do. Rice blast disease Piricularia oryzae. Blasticidin. Cercospora leaf spot of Oarcospora beticola Maneb.

sugar beets. Septoria leaf spot of celery-. Septoria apiigraveolentis... Do. Apple scab Venturia inaeqaalis. Cyprex. Common bacterial blight Xanthomonaa phaseoli.. Streptomycin of bean. sulfate.

Wherever possible, the Applicants recommend the use of in vivo procedures to test the 5-(5'-barbiturilidene)- 4-oxotl1iazolidine-Z-imino HCl salt compositions of this invention to demonstrate their eflicacy under more realistic conditions. However, not all pathogens lend themselves to such techniques. In order to provide additional spectrum definitions, the following fruit-rotting, storage decay In their plant protection aspects, the 5-(5'-barbiturilidene)-4-oxothiazolidine-Z-imino HCl salt of this invention may be used in the manner known to the crop protection art; that is, they can be made up in solid or liquid formulations. Examples of solid formulations are dust, wettable powders, granules and pellets. As a dust, S-(Sbarbiturilidene)-4-oxothiazolidine-2-imino HCl salt compound may be dispersed in powdered solid carriers such as talc, soaps, soapstone, attapulgus clay as well as other finely divided solids known in the dusting art. When formulated as wettable powders, the active component may be employed in conjunction with inert fillers which may be of the clay type carrier or non-clay type, in conjunction with various combinations of wetting agents and emulsifiers which permit the adaptation of the concentration as a freeflowing powder for dispersion in the field..

Each of these carriers may in turn contain other carriers or extenders which are ordinarily non-reacting or inert substances such as sand, clays, talc, sawdust, alkaline earth carbonates, oxides, phosphates and the like as well as diatomaceous earth, micas or other suitable materials. When liquid formulations are desired, liquid extenders, dilutants or carriers of a non-reactive nature may be utilized. Examples of such materials are alcohols, ketones, glycols, aromatic hydrocarbons, petroleum fractions such as octane and various other distillates. From these considerations, it will also be recognized that the above formulations with slight modifications may be used in the field of animal husbandry as dusting powders and salves.

Where it is desired to use the aforementioned wettable powders or liquid formulations, either emulsified, dispersed or suspended in water or other fluids, one or more of the class of materials herein referred to as adjuvants can also be incorporated into the powder, dust or liquid formulation. These adjuvants comprise surface active agents, detergents, wettable agents, stabilizers, dispersing agents, suspending agents, emulsifying agents, Spreaders, stickers and conditioning agents generally. To their modifying characteristics these adjuvants may facilitate handling and application and infrequently enhance or potentiate the (Sbarbiturilidene)-4-oxothiazolidine-2-imino HCl salt compositions of this invention in their biological activities by mechanisms which are frequently not well understood. A satisfactory but not not exhaustive list of these adjuvants appears in Soap Chemical Specialties, vol. 31, No. 7, p. 61; No. 8, pp. 286l; No. 9, pp. 52-67; and No. 10, pp. 38-67 (1955). See also, Bulletin No. 607 of the Bureau of Entomology and Plant Quarantine of the United States Department of Agriculture.

An additional advantage of S-(Sbarbiturilidene)-4-oxothiazolidine-Z-imino HCl salt is its compatibility with a variety of other biocidal and fungicidal materials. For example, it may be convenient to combine S-(Sbarbiturilidene)-4-oxothiazolidine-2-imino HCl salt with one or more other adjuvants, carriers, pesticides, biocides or fungicides of various structures. For example, 5-(5'barbiturilidene)- 4-oxothiazolidine-2-imino HCl fungicidal inhibitors may be combined with insecticidal materials such as chlordane, benzene hexachlorides, DDT, DDD, the insecticidal carbamates, polychlorinated terpenes, parathions, methozychlor, insecticidal phosphates phosphorothioates, phosphorodithioates and with fungicides such as sulphur, quinones, dodecylguanidine and metal dimethyldithiocarbamates.

There are many other considerations such as concentration and method of application which may make some methods of application more favored than others. These considerations may include the type of organisms on which the compound is to be administered, the degree of activity,

. the degree of activity toward the particular organism, and

side effects. Also to be considered is the cost of production and the characteristic solubility of S-(S'barbiturilidene)-4-oxothiazolidine-Z-imino HCl salt in the carrier material.

What is claimed is:

1. A method of killing or inhibiting the growth of microorganisms selected from the group consisting of fungi, Gram positive bacteria and Gram negative bacteria which comprises contacting said microorganisms with 5-(5-barbiturilidene)-4-oxothiazolidine-Z-imino HCl salt in an amount effective to kill or inhibit the growth of said microorganisms.

2. The method according to claim 1 wherein the Gram positive bacteria are selected from the group consisting of Staphylococci, Corynebacter, Listeria, Micrococci, Mycobacterium, Diplococci and Steptococci.

3. The method according to claim 2 wherein the Staphylococci is Staphylococcus aureus.

4. The method according to claim 2 wherein the Corynebacter are selected from the group consisting of Corynebacterium diplztlzeriae and Corynebacterium haemolyticum.

5. The method according to claim 2 wherein the Listeria is Listeria monocytogenes.

6. The method according to claim 2 wherein the Micrococci are selected from the group consisting of Micrococcus tetragena, Micrococcus melitensis and Micrococcus lysodeiktz'cus.

7. The method according to claim 2 wherein the Mycobacterium are selected from the group consisting of Mycobacterium avium, Tuberculosis var. liominis, Mycobacteritlm phlei, Mycobacterium fortuitum, and Mycobacterium butyricum.

8. The method according to claim 2 wherein the Diplococci are selected from the group consisting of Diplococcus intracellularz's and Diplococcus pneumoniae.

9. The method of claim 2 wherein the Streptococci are selected from the group consisting of Streptococcus hemolytic group A, and Streptococcus hemolytic group B.

10. The method according to claim 1 wherein the Gram negative bacteria are selected from the group consisting of Escherichia, Shigella, Salmonella, Vibrio, Neisseria, Haemophilus, Brucella, Proteus, Pseudomonas, Erwinia and Xanthomonas.

11. The method of claim 10 wherein the Xanthomonas is Xantliomonas phaseoli.

12. The method according to claim 10 wherein the Escherichia is Escherichia coli.

13. The method according to claim 10 wherein the Shigella are selected from the group consisting of Shigella boydii, Shigella sonnei and Shigella dysenteriae.

114. The method according to claim 10 wherein the Salmonella are selected from the group consisting of Salmonella derby, Salmonella enteritis, Salmonella gallinarium, Salmonella panama, Salmonella paratyplti, Salmonella pullorum and Salmonella typhosa.

15. The method according to claim 10 wherein the Vibrio are selected from the group consisting of Vibrio cholerae and Vibrio fetus.

16. The method according to claim 10 wherein the Neisseria are selected from the group consisting of Neisseria gonorrhoeae, Neisseria intracellularis and Neisseria meningitides.

17. The method according to claim 10 wherein the Hemophilus are selected from the group consisting of Hemophilus hemolyticus, Hemophilus influenzae, Hemophilus parainfluenzae, Hemophilus suis and Hemophilus vaginalis.

18. The method according to claim 10 wherein the Brucella are selected from the group consisting of Brucella abortus, Brucella melitensis and Brucella suis.

19. The method according to claim 10 wherein the Proteus are selected from the group consisting of Proteus vtllgaris, Proteus morganii and Proteus mirabilis.

20. The method according to claim 10 wherein the pseudomonas are selected from the group consisting of Pseudomonas aeruginosa and Pseudomonas maltophilia.

21. The method according to claim 10 wherein the Erwinia is Erwinia carotovora.

22. The method according to claim 1 wherein the fungi are selected from the group consisting of Fusarium, Penicllium, Aspergillus, Alternaria, Rhizopus, Candida, Rhodotorula, Monascus, Scopulariopsis Cephalosporium, Phoma, Paecilomyces, Nigrospora, Absidia, Thamnidium, Phycomyces, Beauvaria, Phialophora, Cladosporium, Torula, Monosporum, Verticillium, Trichophyton, Cercospora, Pythium, Helminthosporium and Microsporum.

23. The method according to claim 22 wherein the Fusarium is selected from the group consisting of Fusariutn roseum, Fusarium oxysporum and Fusarium oxysporum cubense.

24. The method according to claim 22 wherein the Penicillium is selected from the group consisting of Penicillium rubrum and Penicillin/n notatum.

25. The method according to claim 22 wherein the Aspergillus is selected from the group consisting of Aspergillus niger, Aspergillus sydowi, Aspergillus nidulons, Aspergillus fla'vus, Aspergillus amstelodami and Aspergillus fumigatus.

26. Th method according to claim 22 wherein the Alternaria is Alternaria solani.

27. The method according to claim 22 wherein the Rhizopus is Rhizopus stolonifer.

28. The method according to claim 22 wherein the Candida is selected from the group consisting of Candida albicans, Candida krusei, Candida quilliermondii, Candida tropicalis, Candida pseudotrop'icalis, Candida pulcher-rima, Candida intermedia and Candida zeylanoides.

29. The method according to claim 22 wherein the Rhodotorula is Rhodotorula sp. Dulge.

30. The method according to claim 22 wherein the Monascus is Monascus purpwrea.

31. The method according to claim 22 wherein the Scopulariopsis is Scopulariopsis sp. U.

32. The method according to claim 22 wherein the Cephalosporium is selected from the group consisting of Cephalosporium acremonium and Cephalosporium sp. 0U.

33. The method according to claim 22 wherein the Phoma is Phoma pigmentovora.

34. The method according to claim Paecilomyces is Paecilomyces varioti.

35. The method according to claim 22 Nigrospora is Nigrospora sphaerica.

36. The method according to claim Absidia is Absidia spinosa.

37. The method according to claim Mucor is Mucor racemosus.

38. The method according to claim Thamnidium is Thamnidium elegans.

39. The method according to claim Phycomyces is Phycomyces nitens.

40. The method according to claim 22 wherein the wherein the 22 wherein the 22 wherein the 22 wherein the 22 wherein the 11 wherein the Beauvaria is selected from the group consisting of Beauvaria bassiana and Beauvaria tenella.

41. The method according to claim 22 Phialophora is Phialophora verrucosa.

42. The method according to claim 22 Cladosporium is Cladosporium trichoides.

43. The method according to claim 22 Torula is T orula bergeri.

44. The method according to claim 22 Monosporum is Monosporum apiospermum.

45. The method according to claim 22 Verticillium is Verticillium albo-atrum.

46. The method according to claim 22 wherein the Trichophyton are selected from the group consisting of Trichophyton mentagrophytes and Trichophyton tonsurans.

47. The method according to claim 22 wherein the Cercospora is Cercospora beticola.

48. The method according to claim 22 wherein the Pythium is Pythium arrhenomanes.

49. The method according to claim 22 wherein the Helminthosporium is Helminthosporium oryzae.

50. The method according to claim 22 wherein the Microsporum is M icrosporum gypseum.

wherein the wherein the wherein the wherein the wherein the References Cited UNITED STATES PATENTS 3,464,990 9/1969 Brossi et al. 260257 3,547,937 12/1970 Diana 260302 ALBERT T. MEYERS, Primary Examiner L. SCHENKMAN, Assistant Examiner U.S. Cl. X.R. 71-66; 260258 

1. A METHOD OF KILLING OR INHIBITING THE GROWTH OF MICROORGANISMS SELECTED FROM THE GROUP CONSISTING OF FUNGI, GRAM POSITIVE BACTERIA AND GRAM NEGATIVE BACTERIA WHICH COMPRISES CONTACTING SAID MICROORGANISMS WITH 5-(5''-BARBITURILIDENE)4=OXOTHIAZOLIDINE-2-IMINO HCL SALT IN AN AMOUNT EFFECTIVE TO KILL OR INHIBIT THE GROWTH OF SAID MICROORGANISM. 